Glycosylation of recombinant adeno-associated virus serotype 6

Glycosylation of biopharmaceuticals can affect their safety and efficacy. Glycans can occur on recombinant adeno-associated viruses (rAAVs) that are used for gene therapy; however, the types of glycans that attach to rAAVs are controversial. Here, we conducted lectin microarray analyses on six rAAV serotype 6 (rAAV6) preparations that were produced differently. We demonstrate that O-glycans considered to be attached to rAAV6 were recognized by Agaricus bisporus agglutinin (ABA) and that N-glycans were detected in rAAV6 purified without affinity chromatography. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis showed that the N-glycans detected in rAAV6 were derived from host cell proteins. A combination of ABA-based fractionation and LC-MS/MS revealed that rAAV6 was O-glycosylated with the mucin-type glycans, O-GalNAc (Tn antigen), and mono- and di-sialylated Galβ1-3GalNAc (T antigen) at S156, T162, T194, and T201 in viral protein (VP) 2 and with O-GlcNAc at T242 in VP3. The mucin-type O-glycosylated rAAV6 particles were 0.1%–1% of total particles. Further physicochemical and biological analyses revealed that mucin-type O-glycosylated rAAV6 had a lower ratio of VP1 to VP2/VP3, resulting in a lower transduction efficiency both in vitro and in vivo compared with rAAV6 without mucin-type O-glycans. This report details conclusive evidence of rAAV glycosylation and its impact on rAAV-based therapeutics.


DRVITT(HexNAc)STRTWALPTYNNHLYK
The signal at 40.DPQPLGEPPAT(HexNAcHexNeuAc2)PAAVGPT(HexNAc)TM(oxi)ASGGGAPM(oxi)A  form was abundant for rAAV6 digested without the S-trap digestion, whereas the ratio of mono-and di-oxidized form to non-oxidized form increased after the S-trap digestion.We therefore suspect that some reagent used in the S-trap digestion may accelerate the oxidation of methionine.

Figure S1 .
Figure S1.(A) Western blots of Samples 1-5 detected with anti-hM2BP antibody (left) and anti-AAV viral proteins antibody (right).(B) Western blot analysis of Samples 4 and 5 detected with anti-hM2BP antibody with and without sialidase treatment.(C) The CID mass spectra of hM2BP A64-R76 modified with HexNAc6Hex7FucNeuAc2 in Sample 4.

Figure S3 .
Figure S3.The signals detected by lectin microarray for 2.5 × 10 9 vg rAAV6 samples as described for western blotting.The area of ABA signals is marked by in a red box.The Blank array shows the high signals of Lycopersicon esculentum lectin (LEL), Solanum tuberosum lectin (STL), and Urtica dioica lectin (UDA) in triplicate spots caused by binding to the detection antibody.The signals of those lectins were therefore excluded from the lectin microarray analysis.

Figure S5 .
Figure S5.The experimental workflow of LC-MS/MS analysis for the bound fraction of rAAV6 using Sample 1.

Figure S8 .
Figure S8.The HCD mass spectra of the signals at 40.0 min and 40.8min for D156-G177 modified with HexNAc.The signals m/z 138 and m/z 144 showed similar intensities.

Figure S10 .
Figure S10.XICs of D184-A212 modified by non-(m/z 882.7561 ± 0.005; top), mono-(m/z 882.0878 ± 0.005; middle), and di-oxidized form (m/z 893.4194 ± 0.005; bottom) in (A) the ABA-bound fraction of the peptides digested from ABA-bound rAAV6 with the S-trap column, (B) the peptides digested from ABA-bound rAAV6 without the S-trap column, and (C) the peptides digested from ABA-bound rAAV6 with the S-trap column.The non-oxidized

Figure S11 .
Figure S11.The HCD mass spectra of the signals at 59.2 min and 61.6 min for D184-A212 modified with HexNAc2HexNeuAc.The signals m/z 138 and m/z 144 showed similar intensities.

Figure S12 .
Figure S12.(A) rAAV6 genomes per mouse diploid genome (vg/dg) and (B) hFIX mRNA levels in liver tissue were determined using qPCR 8 weeks after rAAV6 administration.Individual points are shown in black circles, and error bars shows the SD value for n = 4.

Table S1 .
Sample information of the lectin microarrays used for the PCA analysis shown in Figure1B.The number and color correspond to the dot in PCA score plot.

Table S2 .
The list of identified proteins contaminating Sample 4.Several bovine serum proteins (colored blue) and human galectin 3-binding protein (colored red) were identified in Sample 4. )ATQALGR